Fanconi Anemia (FA) is an autosomal recessive cancer susceptibility disorder characterized by multiple congenital abnormalities, progressive bone marrow failure, and cellular sensitivity to DNA crosslinking agents. FA is characterized by hematopoietic stem cell dysfunction, and the treatment of choice for the disease is an allogeneic bone marrow transplant. Since most FA patients do not have a suitable donor, FA is also a candidate disease for gene therapy. The purpose of the current project is to focus on various of the basic biology of FA related to our ongoing gene therapy trials. The specific aims for the five year study period are (1) To develop and characterize a mouse model for Fanconi Anemia Complementation Group G (FANCG knock-out mouse). Our recent studies demonstrate that FA-G patients have more severe disease than FA-A patients. Therefore, a mouse model for FA-G may demonstrate distinct pathology, compared to the previously describe FA- C mouse. The subaims of this section are to generate a colony of FANCG (-/-, -/+, and +/+) mice, to characterize the hematopoietic system in the FANCG knock-out mouse, to characterize the stem cell population in FANCG knock-out, and to perform competitive repopulation studies, comparing the relative engraftment of FANCG (-/-) cells versus FANCG (+/+) cells. In specific aim 2, we will further develop murine models for Fanconi Anemia Gene Therapy in order to compare the transduction of bone marrow cells from FANCC, FANCA, and FANCG knockout mouse models with either pMMP (murine) retroviral vectors or lentiviral vectors. A significant strength of this specific aim will be our ability to assay infection efficiency of transduced bone marrow, by using PCR of cDNA integrations and immunofluorescence of expressed heterologous FA proteins. In specific aim 3, we will systematically compare cell lines and primary cells derived from patients from all nine complementation groups of FA for distinct cellular abnormalities. This aim will be closely aligned with our new Fanconi Anemia Diagnostic and Clinical Center at the DFCI, which has provided us with the opportunity to collect and subtype cell lines from many FA patients. We will compare the ionizing radiation (IR) sensitivity and bleomycin sensitivity of all FA complementation groups, and will examine these cell lines for defects in Non-Homologous End joining (NHEJ) and Homologous Recombination (HR).